Ethanol Inhibits Dopamine Uptake and Augments Rewarding Effects of Cocaine via Organic Cation Transporter 3

Abstract

Concurrent use of cocaine and alcohol is a major cause of emergency hospitalization, underscoring a vital need to understand the mechanistic basis of this highly addictive and dangerous drug combination. Both cocaine (blocker of dopamine (DA), norepinephrine and serotonin transporters, DAT, NET and SERT, respectively) and ethanol (EtOH) increase extracellular levels of these monoamines. However, we and others find no evidence for EtOH interacting with DAT, NET, or SERT, suggesting that EtOH may be acting elsewhere to inhibit uptake of these monoamines. Organic cation transporter 3 (OCT3) is emerging as a key player in the regulation of monoamine signaling. Whether EtOH acts at OCT3 to increase extracellular levels of monoamines is unknown. To this end, we first conducted studies to determine the ability of EtOH to inhibit uptake of [3H]MPP+ into cultured HEK293 cells stably overexpressing human OCT3. We found the IC50 to be 4 mM, showing that concentrations of EtOH reaching brain following behaviorally relevant doses may be sufficient to engage OCT3. We also found that cocaine does not have activity at OCT3 (Mayer et al., 2018, PMID: 29773909), raising the possibility that EtOH may interact with OCT3 to inhibit monoamine uptake, potentially increasing the addictive properties of cocaine and propagating the concurrent use of these drugs. Next, we assessed the effect of EtOH on DAT, NET, and SERT mediated uptake in real‐time by microsuperfusing fluorescent substrate (APP+ or ASP+) onto HEK293 cells stably expressing DAT, NET, or SERT (APP+) or OCT3 (ASP+) before the addition of EtOH. We found that while EtOH did not affect APP+ uptake by DAT, NET, or SERT, it did inhibit accumulation of ASP+ via OCT3 in a concentration‐dependent manner. To further parse out the relationship between EtOH and OCT3, we used high‐speed chronoamperometry to interrogate the effects of local application of EtOH, cocaine, and their combination on clearance of exogenously applied DA from extracellular fluid in striatum of male and female OCT3+/+ and constitutive OCT3‐/‐ mice in vivo. EtOH inhibited DA clearance and enhanced the ability of cocaine to inhibit DA clearance in both male and female OCT3+/+ mice, effects that were lost in OCT3‐/‐ mice. Finally, we conducted behavioral experiments to assess conditioned place preference (CPP) in response to administration of EtOH, cocaine, or both, to determine if coadministration of EtOH and cocaine enhanced place preference compared to administration of each drug individually. The combination of EtOH (1000 mg/kg) and cocaine (3.2 mg/kg), doses which by themselves did not induce CPP, produced robust CPP. CPP studies in OCT3‐/‐ mice are ongoing (n = 7M & 7F/dose) but so far demonstrate no development of CPP, regardless of treatment. Taken together, results suggest that potentiation of the neurochemical and behavioral effects of cocaine by EtOH are OCT3 dependent. OCT3 may be a putative target for therapeutic intervention in the treatment of EtOH and cocaine co‐abuse.